Pesticidal plant extract containing loline derivatives

ABSTRACT

This invention relates to a pesticidal compositions containing at least one pyrrolizidine alkaloid compound derived from a plant and endophyte combination, and applying the pesticidal compositions to another plant without pesticidal protection, where upon application of the composition, the plant confers pest protection. The pyrrolizidine alkaloid compound is of Formula (I) wherein: R═H or CH 3  and R′═H, CH 3 , CHO, COCH 3 .

RELATED APPLICATIONS

This application is a U.S. National Phase of International ApplicationNo. PCT/NZ2008/000052, filed Mar. 12, 2008, designating the U.S. andpublished on Sep. 18, 2008 as WO 2008/111861, which claims priority toNew Zealand Patent Application No. 553892, filed Mar. 15, 2007. Thecontent of these applications is incorporated herein by reference in itsentirety.

STATEMENT OF CORRESPONDING APPLICATIONS

This application is based on the provisional specification filed inrelation to New Zealand Patent Application Number 553892, the entirecontents of which are incorporated herein by reference.

TECHNICAL FIELD

The invention relates to alkaloid based pesticidal composition. Morespecifically the invention relates to a product and methods of providingplants with improved pest protection using naturally producedpyrrolizidine alkaloids.

BACKGROUND ART

Pesticides are useful in protecting plants from attack by various pestsincluding insects. A disadvantage of many pesticides currently marketedare the (perceived or otherwise) issues associated with chemicals andenvironmental concerns. Another problem is that the chemicals requirespecial handling and may be poisonous or harmful to the person applyingthe chemical. Ideally, pesticides would be common-place, have no orminimal environmental effects and utilise protection mechanisms alreadypresent in nature referred to herein as ‘naturally’ produced.

Alkaloids, including pyrrolizidine alkaloids, are produced by endophytesas a fungal metabolite when in symbiotic relationships with plantspecies including grasses. Such endophytes are valued in grasses due tothe pest protection the alkaloids produced from the endophytes provide.In effect, the endophyte provides the plant with a natural built in pestprotection.

Typically, transferring resistance to another plant e.g. an alternativegrass cultivar, has been carried out by infecting the plant with theendophyte. Examples of this process are described in other patentsincluding those pursued by the applicant. A further example is U.S. Pat.No. 7,037,879 which teaches of a method of conferring pest resistance toplants of Poaceae by adding an isolated endophytic bacterium to thePoaceae plant. No teaching is made other than to transfer an endophytesimilar to existing methods of transferring endophytic propertiesamongst grass cultivars. Inoculation may not always be easy or evenpossible between different plants. In addition, the pesticidalproperties desired may not also transfer to other plants whereinoculation is successful. It should also be appreciated that aninoculation step requires careful, slow and comparatively expensivetechniques.

U.S. Pat. No. 6,372,239 teaches of a composition containing a ‘cocktail’of plant alkaloids used as an insecticide. Alkaloids described includeanabasine along with a wide variety of other alkaloids from a variety ofplants. There is no teaching regarding the alkaloids being endophytemetabolites or the alkaloids being loline type compounds. Theformulations described also utilise strong polar solvents with apreferred solvent being mineral turpentine. Such solvents areundesirable due to their cost and environmental impacts.

U.S. Pat. No. 5,185,028 teaches of synthetically produced N-substitutedloline derivative compounds and their use as a pesticide in sprayingapplications. The specification teaches away from use of naturallyderived alkaloid compounds claiming synthetic compounds with a differingC₄ to C₂₀ R₁ group to naturally occurring pyrrolizidine alkaloidcompounds. The specification also teaches that the synthetic lolinederivative be mixed with strong solvents to form a liquid which, asnoted above is not desirable. Further, the specification teaches thatthe solution should be applied at the locus for pesticidal effects suchas by spraying on a leaf. Indirect methods of applications are nottaught or contemplated in the specification.

Yates et al (1990)¹ describes an experiment undertaken to determine thetoxicity of tall fescue extracts where the tall fescue was infected withAcremonium coenophialum endophyte. Yates does not teach or suggestapplying the extract to another plant in order to transfer pesticidalproperties. Yates also does not attribute pesticidal properties topyrrolizidine alkaloids.

Yates et al., ‘Assay of Tall Fescue Seed Extracts, Fractions andAlkaloids Using the Large Milkweed Bug’, J. Agric. Food Chem. 37:354-357(1989).

US 2004/0141955 teaches of a novel endophytic fungi termed ‘Muscodor’which is used to confer pest resistance to plants by inoculation ofMuscodor species into the plant. A further option is described being useof a stabilised Muscodor placed adjacent or near the plant to beprotected and the volatile compounds produced by the Muscodor providethe pesticidal effect. No disclosure is made regarding pyrrolizidinecompounds, extracts of these compounds, or introducing these compoundsinto a plant. Pesticidal properties are only attributed to the Muscodorendophyte.

Casabuono et al 1997 teaches that loline alkaloids of Festuca argentinawere asymptomatic and non-toxic at dose ranges from 31.25 to 125.0 mg/kgbased on studies where such alkaloids were isolated and administered tomice as a concentrated aqueous suspension. No teaching is made withinthe paper regarding use of the isolated loline alkaloids as a pesticide.Of interest though is that loline alkaloids, whilst having a pesticidalaffect, do not appear to be particularly toxic which may be useful inhorticultural applications of the present invention.

From the above discussion, it should be appreciated that it would bedesirable to have a pesticidal composition that utilised pesticidalprotection mechanisms present in nature but without the need forexample, to inoculate the plant with an endophyte.

It is an object of the present invention to address the foregoingproblems or at least to provide the public with a useful choice.

All references, including any patents or patent applications cited inthis specification are hereby incorporated by reference. No admission ismade that any reference constitutes prior art. The discussion of thereferences states what their authors assert, and the applicants reservethe right to challenge the accuracy and pertinency of the citeddocuments. It will be clearly understood that, although a number ofprior art publications are referred to herein, this reference does notconstitute an admission that any of these documents form part of thecommon general knowledge in the art, in New Zealand or in any othercountry.

It is acknowledged that the term ‘comprise’ may, under varyingjurisdictions, be attributed with either an exclusive or an inclusivemeaning. For the purpose of this specification, and unless otherwisenoted, the term ‘comprise’ shall have an inclusive meaning—i.e. that itwill be taken to mean an inclusion of not only the listed components itdirectly references, but also other non-specified components orelements. This rationale will also be used when the term ‘comprised’ or‘comprising’ is used in relation to one or more steps in a method orprocess.

Further aspects and advantages of the present invention will becomeapparent from the ensuing description which is given by way of exampleonly.

DISCLOSURE OF INVENTION

Compositions and methods are now described relating to the inventorsunexpected finding that natural pesticidal effects observed in grass andendophyte combinations may be transferred to other plants without needto inoculate endophytes into plants on which the benefits are to beconferred.

For the purposes of this specification, the term ‘composition’ refers toone or more compounds in a close association.

The term ‘pesticidal’ refers to a composition for deterring ordestroying plant, fungal, or animal pests including insects.

According to one aspect of the present invention there is provided apesticidal composition obtained from an aqueous extraction of a plantand endophyte combination, wherein the aqueous extract contains at leastone pyrrolizidine alkaloid compound, wherein the pyrrolizidine compoundis of Formula [I] being:

wherein:

-   -   R═H or CH₃ and    -   R′═H, CH₃, CHO, COCH₃.

According to a further aspect of the present invention there is provideda plant with enhanced pest protection characterised in that the plantconfers said pest protection upon application of a pesticidalcomposition wherein the pesticidal composition includes at least onepyrrolizidine alkaloid compound of Formula [I]:

wherein:

-   -   R═H or CH₃ and    -   R′═H, CH₃, CHO, COCH₃;        and further characterised in that the plant is not infected with        an endophyte.

According to a further aspect of the present invention there is provideda method of producing a pesticidal composition by the steps of:

-   (a) cultivating at least one plant or part thereof inoculated with    at least one endophyte and,-   (b) extracting by way of aqueous extraction processes at least one    pyrrolizidine alkaloid compound from the plant or part thereof to    produce the pesticidal composition.

According to a further aspect of the present invention there is provideda method of conferring pest resistance to at least one plant by thesteps of:

-   (a) producing a pesticidal composition from a natural source    containing at least one pyrrolizidine alkaloid compound; and,-   (b) applying the pesticidal composition to the medium in which the    plant is growing allowing the plant to absorb the composition via    the plants roots.

According to a further aspect of the present invention there is provideda method of conferring pest protection to at least one plant by thesteps of:

-   (a) producing a pesticidal composition from a non-synthetic source    containing at least one pyrrolizidine alkaloid compound; and,-   (b) applying the pesticidal composition to a plant on which    increased pest protection is to be conferred.

According to a further aspect of the present invention there is provideda method of conferring pest protection to at least one plant by thesteps of:

-   (a) cultivating at least one plant or part thereof inoculated with    at least one endophyte;-   (b) extracting at least one pyrrolizidine alkaloid compound from the    plant or part thereof to form a pesticidal composition and;-   (c) applying the composition to a plant on which increased pest    protection is to be conferred.

According to a further aspect of the present invention there is providedthe use of a pesticidal composition in conferring pest resistance to atleast one plant wherein the composition contains at least onepyrrolizidine alkaloid compound derived from a plant and endophytecombination with the structure of Formula [I] being:

wherein:

-   -   R═H or CH₃ and    -   R′═H, CH₃, CHO, COCH₃.

According to a further aspect of the present invention there is providedthe use of a plant inoculated with an endophyte to produce a pesticidalcomposition containing at least one pesticidally effective pyrrolizidinealkaloid compound.

According to a further aspect of the present invention there is providedthe use of an endophyte producing at least one pyrrolizidine alkaloidcompound when in combination with a grass cultivar, to confer pestresistance to a plant that is not infected with an endophyte.

In one embodiment, the pesticidal composition includes at least onepyrrolizidine alkaloid compound produced from an endophyte and grasscombination.

Preferably, the pyrrolizidine alkaloid compound or compounds is a1-aminopyrrolizidine compound. More preferably, the pyrrolizidinealkaloid compound or compounds are as per the structure of Formula [I]being:

wherein:

-   -   R═H or CH₃ and    -   R′═H, CH₃, CHO, COCH₃.

More specifically, Formula [I] includes:

-   -   Ioline where R═CH3 and R′═H;    -   norloline where R═H and R′═H;    -   N-methylloline where R═CH3 and R′═CH3;    -   N-formylloline where R═CH3 and R′═CHO;    -   N-formylnorloline where R═H and R′═CHO;    -   N-acetylloline where R═CH3 and R′═COCH3;    -   N-acetylnorloline where R═H and R′═COCH3.

Preferably, the plant or part thereof is derived from the genus Festuca.More preferably, the plant or part thereof is a meadow fescue or tallfescue species grass. For the purposes of this specification, referencewill now be made to the plant or part thereof being derived from a grassplant. This should not be seen as limiting as it should be appreciatedthat other plants also are inoculated by endophytes which producealkaloid compounds.

The term ‘plant or part thereof’ as used herein refers to the entireplant or seeds, roots, leaves, flowers, stems, pseudo-stems and thelike. In the present invention, seeds have been found to be the mostpreferable source for extracting pyrrolizidine alkaloid compoundsalthough, other plant parts (or the whole plant) may be used. It shouldbe appreciated that the plant part used may be dependent on where thegreatest levels of pyrrolizidine alkaloid compounds are produced andalso dependent on extraction techniques used.

As may also be appreciated, the part of the plant used may also varyseasonally as well as for example, total alkaloid levels per plantincrease in spring, reaches a peak at seed maturation, drops quicklywith seed dispersal and stalk senescence and increases again duringvegetative growth in late summer.

Preferably, the endophyte used is characterised by producing at leastone pyrrolizidine alkaloid compound. More preferably, endophytes includethose of the genera Epichloe or Neotyphodium.

As noted above, the base grass plant is infected with endophytic fungi.Such combinations are well known in the art where the endophyte providesa symbiotic relationship with the grass and provides pest resistance tothe infected grass.

Preferably, the plant to which the pesticidal composition is applieddevelops resistance to pests akin to that observed for a grass andendophyte combination but the plant does not include the endophyte. Asmay be appreciated, inoculation with an endophyte is a specialisedprocess and a process that does not work for all plant species.Inoculation, if successful may also result in different properties, notnecessarily pesticidal related. The pesticidal advantages of theendophyte are very useful hence the composition and method of thepresent invention allows transfer of the beneficial properties of theendophyte without the need to go through an inoculation process.

In one embodiment the resistance produced in the plant is systemic withthe pesticidal composition being absorbed and circulated by the plant ororganisms in the plant so as to confer a pest resistance to the plant.

In alternative embodiments the pesticide is a contact pesticide whichdeters pests via volatile production or, on contact with the pest, killsor deters the pest from eating the plant.

An example of this pest resistance mechanism includes direct deterringof pests feeding on the plant via volatile production and/or by a postdigestive feedback mechanism where the pest eats a portion of the plant,ingests toxins and develops a learned response to not eat that plantagain. It is likely that many interactions may occur to cause theobserved resistance and the above example is provided by way ofexplanation and should not be seen as limiting.

Preferably the plant has resistance to both plant shoot feeding and rootsucking and chewing pests. This feature of the present invention isparticularly advantageous as it provides a full spectrum pesticideavoiding the need to use two or more pesticides in order to gain thedesired effects.

More specifically, pest resistance may be developed to at least: grassgrub (Cotelytra zealandica), porina larvae (Wiseana spp), milkweed bug(Oncopeltus fasciatus), aphid spp (Rhopalosiphum padi and Schizaphisgraminum), Japanese beetle, spittle bug and diamond back moth. As shouldbe obvious to persons skilled in the art, other insects known to becontrolled by endophyte and plant associations are also candidates forpest protection according to the invention. The above list is providedby way of example only and should not be seen as limiting.

In one embodiment, a pesticidal composition is produced as an aqueousextract by the steps of grinding and mixing using seeds and water. Asshould be appreciated, seed grinding may occur before addition of waterand mixing or may occur simultaneously with grinding occurring inconjunction with mixing with water. An aim of the extraction step is torelease the pyrrolizidine alkaloid compounds from the seed. Many othermethods are envisaged such as use of other solvents including alcohol inthe extraction or other extraction techniques such as super criticalfluid extraction, pressing, filtration methods and the like. However,water is a simple, inexpensive and environmentally friendly solvent.Also, water is useful in this instance as the pyrrolizidine alkaloidcompounds of interest are sufficiently water soluble to produce usefulamounts of these compounds in the resulting liquid to form an extract orpesticidal composition. The use of water extraction should not be seenas limiting as it should be appreciated that many separation techniquesexist in the art which may be applicable to the present invention.

Preferably, the composition contains a pesticidally effective amount ofat least one pyrrolizidine alkaloid to induce the desired response andis predetermined by routine testing. Where the ultimate response isinsect resistance, an ‘effective amount’ or ‘pesticidally effectiveamount’ is defined to mean those quantities which will result in asignificant resistance to a test group compared to an untreated group.The actual effective amount may vary with plant species and/or thetarget pest species stage of development, environmental conditions,nature of substrate, type of carrier, period of treatment and otherrelated factors.

Based on trials completed by the inventor, a liquid extract pesticidalcomposition using water as the solvent and seeds as the plant partproduces a solution containing effective amounts of pyrrolizidinealkaloids. For example, levels include a loline content of 311-1962 ppm,an N-acetylloline content of 36-354 ppm, an N-acetylnorloline content of7-88 ppm and an N-formylloline content of 268-1520 ppm. Amounts areprovided by way of illustration and the composition may include otheralkaloids beyond those illustrated here. It should also be appreciatedthat alternative extraction methods may have greater extractionefficiency and thereby produce an extract composition containing greater(or lower) amounts of the above identified compounds. It should also beunderstood that other pyrrolizidine alkaloids present in the compositionmay also assist or influence the pesticidal effect.

Also, based on the inventor's experience, it is possible to dilute theconcentration of the alkaloid content in the composition considerablyand still have a beneficial effect. In a preferred embodiment, a lowerthreshold of a 12.5% by volume dilution may be made from a water basedextraction of ground seed and which still includes sufficient levels ofpyrrolizidine alkaloids to confer a pesticidal effect to plants on whichthe solution is applied. This result suggests that the pesticidal effectof the composition is strong and that only minimal amounts may berequired in a final product therefore making the composition attractiveand cost effective commercially.

As noted above, the pesticidal composition is applied to a plant orplants in order to confer pest resistance. Preferably, the deliverymethod or methods include pouring an aqueous solution of the compositiononto the growing medium (e.g. soil, potting mix etc) surrounding theroots of the plant or plants and the solution is taken up into the plantby the plant roots. The mechanism for this is understood to be aleaching process of the composition into the soil and plant uptake vianatural osmotic processes.

Alternative methods of delivery also include spraying a solution ontothe plant; pouring a powder of the composition onto the plant directlyor in the vicinity of the plant roots; soaking or immersion of a plantor part thereof in the composition; dusting the plant above the ground;fumigating the composition e.g. as an aerosol; insertion or placing asolid plug of composition into or adjacent the plant e.g. inserting theplug into a hole drilled into the plant stem or attaching the plugadjacent the plant stem; application as a prill or granule to the mediumin which the plant grows; inserting the composition into a plantirrigation line; inserting the composition into a hydroponic system; andcombinations thereof.

The above methods are provided by way of example only and it should beappreciated that other delivery methods may also be undertaken withoutdeparting from the scope of the invention. For further discussion,reference will be made to the composition being an aqueous solutionwatered onto the plant roots and absorbed into the plant via the plantroots. This should not be seen as limiting.

Plants to which pesticidal resistance may be conferred do not appear tobe limited to any particular species or group of species. The inventorhas found that pesticidal effects may be transferred to other grassessuch as Festuca spp. and Lolium spp., brassicas including cabbage,cereal crops such as barley, horticultural crops such as tomato plants,and flowering crops such as roses. One surprising and useful result ofthe transfer in pest resistance is that the alkaloid compounds do notappear to transfer to edible portions of the plant such as the plantfruit. This may be advantageous in horticultural crops where it would beundesirable, at least from a marketing viewpoint, if the crop includedalkaloid compounds that were ingested by humans.

From the above description it should be appreciate that the inventor hasfound that the pesticidal effects of endophytes in grass may betransferred to another plant without also having to transfer theendophyte. In effect, the symbiotic relationship between a grass andendophyte combination may be transferred to other plants without need toalso inoculate the other plant with endophyte.

Uses of the pesticidal composition and method may include horticulturalapplications (e.g. market gardens, glasshouse crops and so on),viticulture (e.g. to prevent pests in wine producing grapes and grapeplants), agriculture (e.g. to prevent pest predation of forage crops,cereal crops and so on). Addition of the alkaloid compound to dripirrigation or hydroponic systems is also a further usage options.

A further advantage of the present pesticidal composition and method isthat they present an alternative ‘natural’ and renewable remedy againstpests in plants contrary to existing methods of pest control such asusing chemical based sprays such as Shield™ for control of pests inroses.

BRIEF DESCRIPTION OF DRAWINGS

Further aspects of the present invention will become apparent from thefollowing description which is given by way of example only and withreference to the accompanying drawings in which:

FIG. 1 shows a graph comparing aphid numbers on plants after 21 dayswith and without treatment using a pesticidal composition in a cafeteriasituation (Pots were randomised in 5 replicates);

FIG. 2 shows a graph comparing aphid numbers on plants after 27 dayswith and without treatment using a pesticidal composition in a cafeteriasituation (As in FIG. 1);

FIG. 3 shows a graph comparing aphid numbers on nil endophyte fescue andryegrass plants after 31 days with and without treatment using apesticidal composition in a cafeteria situation (As in FIG. 1 aphidswere counted when plants were destructively harvested);

FIG. 4 shows a graph comparing insect numbers on barley plants over timewith and without treatment using a pesticidal composition in a cafeteriasituation (insects were placed in an enclosure and were free to chooseplants);

FIG. 5 shows a graph for varying strength solutions of pesticidalcomposition and the effect they had on mature aphid populations overtime;

FIG. 6 shows a graph scoring relative plant health over time for plantsinfected with mature aphid populations and treated with varying strengthsolutions;

FIG. 7 shows a graph for varying strength solutions of pesticidalcomposition and the effect they had on immature aphid populations overtime; and,

FIG. 8 shows a graph scoring relative plant health over time for plantsinfected with immature aphid populations and treated with varyingstrength solutions.

BEST MODES FOR CARRYING OUT THE INVENTION

The invention is now described with reference to a series of experimentscompleted by the inventor.

Experiments (presented as Examples below) were undertaken to determineseveral aspects being:

-   -   1. Can a pesticidal composition be produced from a        plant/endophyte combination?    -   2. Can the pesticidal composition containing loline compounds be        absorbed through their roots into grass plants that do not        include an endophyte?    -   3. Do the pesticidal properties transfer to the plant?    -   4. Does the composition and method confer pest resistance to        other insects?    -   5. Will treated plants have an effect as a pesticide in a choice        cafeteria situation?    -   6. Does the pesticidal composition transfer to edible parts of        the plant and therefore influence whether or not the food        produced from the plant contains loline compounds?

Example 1

Seed produced from a Meadow Fescue grass and endophyte combination wascollected, ground and then mixed with water. The mixture was thenfurther ground/macerated using a vigorous stirring method that bothmixes and further breaks apart the seed fractions. After thoroughmixing, the resulting solution was allowed to settle with seed husks andother solids separating out to the bottom of the mixing vessel. Theliquid at the top of the vessel was collected, filtered and analysed todetermine that a pesticidal composition was produced and what quantitiesof selected compounds were extracted. The process was repeated fivetimes. A sixth sample was also obtained and analysed from the husks andresidue of Grinding Number 4. This material was found to still containhigh concentrations of loline compounds. Further improvements toextraction techniques are considered obvious and this method is providedby way of example only.

Results found in terms of various loline compounds measured are shownbelow in Table 1.

TABLE 1 Pesticidal Composition Contents Pyrrolizidine Alkaloid Content[mg/kg or ppm] Total of NAL, Grinding Number NANL and NFL NAL¹ NANL²NFL³ 1 311 36 7 268 2 464 75 30 359 3 444 70 27 347 4 597 90 40 467 5423 63 16 344 Residue from 1962 354 88 1520 Grinding Number 4 ¹NALrefers to N-acetylloline ²NANL refers to N-acetylnorloline ³NFL refersto N-formyllolineplant/endophyte seed containing pyrrolizidine alkaloid compounds. Theresults for the extracted residue suggest that further extractionmethods may be possible to extract more pyrrolizidine alkaloids.

Example 2

Pesticidal composition from Grinding Number 1 of Example 1 was wateredonto the roots of endophyte free Fescue and Ryegrass plants grown in aglasshouse under controlled conditions into silica sand filled pots.Control plants were included in the plant samples and were watered withnil endophyte ground extract containing no pesticidal composition. Trialplants were each watered with 100 ml of each composition every secondday with nutrient watered on alternate days. After 21 days, a sub-sampleof leaf material was analysed to determine the uptake of pesticidalcomposition into the plants tested.

The results shown below in Table 2 for endophyte free Fescue plants andTable 3 for endophyte free Ryegrass plants clearly indicate transfer ofthe measured pyrrolizidine alkaloid compounds from the composition andinto the plants via their root system

TABLE 2 Alkaloids in Endophyte Free Fescue Plants Pyrrolizidine AlkaloidContent [mg/kg or ppm] Total of NAL, Plant Number NANL and NFL NAL¹NANL² NFL³ 1 767 119 0 648 2 548 69 0 479 3 577 95 0 482 4 461 56 0 4055 420 38 0 382 Average 555 75 0 479

TABLE 3 Alkaloids in Endophyte Free Ryegrass Plants PyrrolizidineAlkaloid Content [mg/kg or ppm] Total of NAL, Plant Number NANL and NFLNAL¹ NANL² NFL³ 1 1366 214 0 1152 2 828 87 44 697 3 790 84 27 680 4 39240 0 352 5 376 37 0 340 Average 750 92 14 644 ¹NAL refers toN-acetylloline ²NANL refers to N-acetylnorloline ³NFL refers toN-formylloline

Example 3

To test whether or not pest protection transferred to the endophyte freeplants, the plants used in Example 2 were subjected to aphid attack. Dueto there being both treated and untreated (control) plants present inthe same area and under the same growing conditions, the aphids werepresented with a cafeteria situation where they were able to chooseplants to feed on at will and without other factors influencing.

The number of aphids on each plant was measured after 21 days, 27 daysand 31 days post starting the trial with aphids being removed after eachsampling date.

FIGS. 1 to 3 show the results of this trial. As can be seen on thegraphs, aphid numbers were dramatically lower on treated plants asopposed to untreated plants showing a significant pesticidal effect fromapplication of the pesticidal composition. Therefore it is clear thatthe pesticidal composition does have a pesticidal effect.

Example 4

A further trial was completed to determine if the pesticidal effectextends to root feeding insects such as grass grub.

Small samples of carrot were produced and soaked in pesticidalcomposition from Grinding Number 2 in Example 1. Additional controltreatment samples were also tested being fresh carrot, carrot soaked inwater, and carrot soaked in nil endophyte extract. Grass grubs were fedthe various samples in a no-choice arrangement and after 3 days, theamount of carrot eaten was observed and measured.

Observations noted were that carrot soaked with pesticidal compositionwas either not eaten or only slightly eaten. In contrast, controlsamples were completely eaten. Comparative measurements of the grassgrubs tested showed that grubs fed treated carrot showed a weight losscompared to control samples and some grub mortality for treated samplesnot observed in control samples.

The above trial confirms that the pesticidal composition has pesticidaleffects, on root feeding pests including grass grub. Grass grub exposedto the pesticidal material; refused at subsequent feeding to consumefresh untreated carrots; suggesting a post digestive feedback response.

Example 5

A further trial was completed to determine if the pesticidal effectcould be transferred to plants other than those of the Graminae family.

Cabbage brassica were tested with the pesticidal composition of GrindNumber 3 of Example 1 watered onto the pots in which the trial cabbageplants were grown in silica sand.

The results found are shown in Table 3 below.

TABLE 3 Cabbage Treated with Pesticidal Composition PyrrolizidineAlkaloid Content [mg/kg or ppm] Total of NAL, NANL and NFL NAL¹ NANL²NFL¹ 154 6 0 148 ¹NAL refers to N-acetylloline ²NANL refers toN-acetylnorloline ¹NFL refers to N-formylloline

The results found show that transfer occurs into plants other than justgrasses.

Example 6

A further experiment was completed to confirm that the pesticidal effectcould be transferred to further plants. The trial used barley plantstreated with the pesticidal composition of Grinding Number 5 of Example1 alongside control samples which were untreated. The plants were alsosubjected to insect attack in order to explicitly show the pesticidaleffect and to determine if the insects responded in a cafeteriasituation as expected.

As shown in FIG. 4, the pesticidal effect was clearly apparent withinsect numbers dramatically different between treated and untreatedbarley samples. The trial also shows again that the effect may betransferred to plants other than grasses.

Example 7

Barley plants were further tested to determine the concentration atwhich the pesticidal composition could be diluted and still achievepesticidal results

Barley plants were placed in inert material and the plant samples weretreated with varying concentrations of pesticidal composition producedfrom Grinding Number 5 of Example 1. Concentrations were from 100% beingthe composition of Grinding Number 5 and diluted using water toconcentrations of 50%, 25%, 12.5%, and 6%. A control was also used withno treatment composition. A total of 10 aphids were placed on each plantafter treatment (or no treatment for the control) described above.

The effect was tested using both mature and immature aphids (Bird CherryOat Aphids (R. padi) with ten insects placed on each plant on day 1 ofthe trial. Mature insects would be expected to multiply quickly placingmore stress on the plant whereas immature insects would be expected tohave a lag effect before multiplying.

As can be seen in FIG. 5, mature insects multiplied as expected forconcentrations lower than 25% but for concentrations of 12.5% or greaterthe multiplication was reduced or completely stopped. FIG. 7 shows theeffect for immature insects which was even more marked with a 12.5%concentration being sufficient to halt insect multiplication.

FIGS. 6 and 8 show observations of the plant health over time once theplants were subjected to insect attack. In FIG. 6, plant healthdecreases rapidly for low concentration treatments. In FIG. 8 the effectis more noticeable with plants remaining healthy for longer as theconcentration of treatment increased. It should be noted in both casesthat one would expect a drop in plant health owing to the plantnaturally depleting the nutrient supply and being subjected to incessantinsect attack.

The above trial shows that the pesticidal effect is strong with adilution to 12.5% by volume still providing pesticidal effects. Withimproved extraction techniques, it should be appreciated that thecomposition is likely to be cost effective due to only low dilutionsrequired to achieve the desired result.

Example 8

A further trial was completed to determine if the pesticidal effectcould be transferred to tomato (Solarium lycopersicum) plants and alsoto determine if the pyrrolizidine alkaloids transfer into the fruitand/or leaves of the plant.

30 tomato plants were grown under controlled conditions and eithertreated with the solution of Grinding Number 4 of Example 1 or leftuntreated (control).

Once the plants had grown to a stage where multiple green fruit hadappeared and were

maturing, samples were taken to determine the loline compound levels.Loline compounds were detected in tomato plant stems (15 to 25 ppm) butno loline compounds were detected in the leaves or plant fruit.

The trial shows that it is unlikely that pesticidal composition used inthe present invention will carry through into the human food chain bytransfer of pesticidal compounds into leaves of fruit produced by theplant used for edible products.

Example 9

A further trial was completed using the seed residue from GrindingNumber 4 of Example 1. As noted in Example 1, the pesticidal compoundsmeasured in the residue were still considerable.

The residue was placed around the roots of rose plants in theenvironment that had not previously been treated for pests andobservations made on a regular basis to look for any visual signs ofinsect attack.

In the time period observed (over 20 days), no observations were made ofinsect attack and the roses remained free of pests such as aphids. Giventhe amount of chemical sprays used on roses to control pests, thequalitative results found in this Example are at least promising as analternative pest treatment method.

Example 10

Other delivery methods are described.

In one example, the pesticidal compositions produced in Example 1 aredried and formed into a powder. The powder is then spread around theroots of the plant and watered into the ground. Alternatively, thepowder may be compressed into a solid plug and inserted into or adjacentthe plant stem and released into the plant by normal plant osmoticaction.

The powder or a liquid may also be formulated so as to release thepesticidal composition in a slow or fast manner as used in manyfertiliser applications. As an example, one type of slow releasefertiliser is a product named Osmocote™ which may be a granular productthat releases fertiliser in a slow manner. The pesticidal composition ofthe present invention may be added to the Osmocote™ to produce a dualpurpose product both feeding a plant and protecting it from pests.

A further option is to spray a liquid solution containing the pesticidalcomposition onto the plant or dust the plant with a powdered pesticidalcomposition. A yet further option is an aerosol containing pesticidalcomposition or the addition of the pesticidal compounds to hydroponic ordrip irrigation systems.

It should be appreciated form the above Examples that there is provideda pesticidal composition containing naturally occurring compounds. Thecomposition has been shown to be useful in transferring the pesticidaleffects normally attributed to endophyte infection from an endophyte andplant combination into a plant that does not have an endophyte. Theresult is that pest resistance is conferred to the plant without needfor inoculation.

Aspects of the present invention have been described by way of exampleonly and it should be appreciated that modifications and additions maybe made thereto without departing from the scope thereof as defined inthe appended claims.

What I claim is:
 1. A method of conferring pest protection to at least afirst plant or part thereof in need thereof comprising: (a) extracting asecond plant, or part thereof, that has been inoculated with at leastone endophyte by grinding the second plant or part thereof, mixing theground second plant or part thereof with water only to provide a waterextract of the second plant or part thereof and filtering the waterextract of the second plant or part thereof to obtain a pesticidalcomposition, wherein said pesticidal composition comprises at least onepyrrolizidine alkaloid compound, wherein said second plant or partthereof is a meadow fescue species grass, and wherein the second plantor plant part is selected from the group consisting of: the entireplant, seeds, roots, leaves, flowers, stems, pseudo-stems andcombinations thereof; and, (b) applying an effective amount of thepesticidal composition to the first plant or part thereof, whereinapplying the pesticidal composition confers insect pest protection tothe first plant or part thereof.
 2. The method of claim 1, wherein theendophyte produces at least one pyrrolizidine alkaloid compound.
 3. Themethod of claim 1, wherein the endophyte is of the genus Epichloe orNeotyphodium.
 4. The method of claim 1, wherein the method furthercomprises an initial step of (a′) cultivating the second plant or partthereof with at least one endophyte, wherein the second plant or partthereof is different from the first plant or part thereof.
 5. The methodof claim 1, wherein the at least one pyrrolizidine alkaloid compound isa 1-aminopyrrolizidine compound.
 6. The method of claim 1, wherein theat least one pyrrolizidine alkaloid compound has a structure of Formula[I]:

wherein: R═H or CH₃ and R′═H, CH₃, CHO, COCH₃.
 7. The method of claim 1,wherein the first plant or part thereof does not include an endophyte;and wherein the first plant or part thereof develops resistance topests, wherein the resistance is systemic resistance; and wherein theresistance is to shoot feeding, root sucking and chewing pests.
 8. Themethod of claim 1, wherein the first plant or part thereof developsresistance to a pest selected from the group consisting of grass grublarvae (Cotelytra zealandica), porina larvae (Wiseana spp), milkweed bug(Oncopeltus fasciatus), aphid species (Rhopalosiphum padi and Schizaphisgraminum), and Japanese beetle larvae after application of thepesticidal composition in b.).
 9. The method of claim 1, wherein saideffective amount of the pesticidal composition is applied by: i.)pouring an aqueous solution comprising the pesticidal composition ontoroots of the first plant or part thereof, wherein the first plant orpart thereof absorbs the pesticidal composition via the roots; ii.)spraying a solution comprising the pesticidal composition onto the firstplant or part thereof; iii.) pouring a powder comprising the pesticidalcomposition directly onto or near roots of the first plant or partthereof; iv.) soaking or immersing the first plant or part thereof inthe pesticidal composition; v.) dusting above ground parts of the firstplant or part thereof with a powder comprising the pesticidalcomposition; vi.) fumigating above ground parts of the first plant orpart thereof with the pesticidal composition; vii.) inserting or placinga solid plug comprising the pesticidal composition into or adjacent tothe first plant or part thereof; viii.) applying a prill or granulecomprising the pesticidal composition to growth medium of the firstplant or part thereof; ix.) delivering the pesticidal composition via aplant irrigation line to the first plant or part thereof; x.) deliveringthe pesticidal composition via a hydroponic system to the first plant orpart thereof; or xi.) applying the pesticidal composition in aqueous,powder or residue form to the soil or growth medium containing the firstplant or part thereof, or to roots of the first plant or part thereof.10. The method of claim 1, wherein the first plant or part thereof towhich pest protection is conferred is selected from the group consistingof: a grass, a brassica, a cereal crop, a horticultural crop, and aflowering crop.
 11. The method of claim 10, wherein the first plant orpart thereof is selected from the group consisting of a Festuca spp.plant, a Lolium spp. plant, a cabbage plant, a barley plant, a tomatoplant and a rose plant.